Development and evaluation of the polymerase chain reaction (PCR) method for diagnosis of spring viremia of carp virus (SVCV)

Aquaculture has been expanded rapidly to become a major commercial and food-producing sector worldwide in recent decade. In parallel, viral diseases rapidly spread among farms causing enormous economic losses. The accurate detection of pathogens at early stages of infection is a key point for disease control in aquaculture. Spring Viraemia of Carp Virus (SVCV) is a very severe pathogen of carp fishes in different parts of the world and is categorized as a reportable listed disease in the annual published list of World Organization for animal Health (OIE). The objective of this study was to develop and evaluate RT- PCR test for detecting SVC virus and also the sensitivity and specificity of this test. A semi nested RT- PCR was designed using combination of three primers: two external (SVCF , SVCR) and one internal (SVCS) primers which based on conserved region of G gen. The specificity of designed primers (only external ones) by examination on Viral Hemorrhagic Septicemia Virus (VHSV) and Infectious Hematopoietic Necrosis Virus (IHNV) was confirmed. For optimizing of the PCR test, primer concentration, primer annealing temperature, cycle number and Mgcl2 concentration were surveyed. Also for validity test, prevention of false negative and Assurance of its accuracy, a competitive internal control (mimic) designed and its suitable concentration was defined. Evaluation of the sensitivity of designed test were conducted first by comparing the different commercially available RNA isolation guidelines, two guidelines: isotiocyanate phenol–chloroform based protocols (RNX–Plus Iran, Iq2000 kit Taiwan ) and two column based protocols (Cinna pure RNA Iran , high pure viral RNA kit, Roche Germany ). The results indicated that the column based protocols (Roche method and Cinna pure), yield 36.77 ng/μl and 16/47 ng/μl RNA concentration respectively, which were significantly higher than other protocols(P<0.05). Then for evaluation of extracted RNA sensitivity, Serial dilution of SVCV strain 56.70 grown in EPC (1.9×105 TCID50/ml) was examined To compare sensitivity. Extracted RNA from serial dilution with stone's primers and commercial IQ-2000 kit were examined simultaneously. The result indicated that designed semi- nested RT- PCR was able to recognize SVC virus to 10-4 dilution and stone's primer recognize to 10-3 dilution whereas Iq-2000 commercial kit did not recognized in any dilution. In high virus titer in designed test two DNA band (462 bp and 266 bp) produced, and by decreasing virus titer 462 bp was omitted. In low virus titer or lack of virus, just DNA band (mimic) 729 bp can propagate. After designing and optimizing PCR test, a total of 400 suspected cultured Cyprinus carpio with high mortality from 4 aquaculture zone of Khuzestan province were collected and tested for SVCV during 2012- 2013 using developed PCR method and IQ- 2000. The results indicated that SVC virus was not observed in samples using both methods.

Endocrine-disrupting compounds: A review of their challenge to sustainable and safe water supply and water reuse

The relevance of endocrine-disrupting compounds as potential contaminants of drinking water is reviewed, particularly in the reuse of wastewater. Growing populations and increasing intensification of land and water use for industry and agriculture have increased the need to reclaim wastewater for reuse, including to supplement the drinking water supply. The variety of anthropogenic chemicals that have been identified as potential endocrine disruptors in the environment and the problems arising from their use as human and livestock pharmaceuticals, as agricultural chemicals and in industry are discussed. The potentially adverse impact of these chemicals on human health and the ecology of the natural environment are reviewed. Data for the removal of estrogenic compounds from wastewater treatment are presented, together with the comparative potencies of estrogenic compounds. The relative exposure to estrogens of women on oral contraceptives, hormone replacement therapy, and through food consumption is estimated. A brief overview of some methods available or under development for the assessment of estrogenic activity in environmental samples is provided. The review concludes with a discussion of the directions for further investigation, which include human epidemiology, methodology development, and wastewater monitoring. (C) 2006 Wiley Periodicals, Inc.

Contribuition of nitrogen from biological nitrogen fixation, nitrogen fertilizer and soil nitrogen on the growth of the common bean and cowpea

Biological nitrogen fixation (BNF) constitutes a valuable source of this nutrient for the common bean Phaseolus vulgaris L and cowpea Vigna unguiculata (L.) Walp., being its avaibility affected by mineral N in the soil solution. The objectives of this work were to evaluate the effects of nitrogen rate, as urea, on symbiotic fixation of N(2) in common bean and cowpea plants, using the isotopic technique, and quantifying the relative contributions of N sources symbiotic N(2) fixation, soil native nitrogen and urea N on the growth of the common bean and cowpea. Non nodulating soybean plants were used as standard. The research was carried out in greenhouse, using pots with 5 kg of soil from a Typic Haplustox (Dystrophic Red Yellow Latosol). The experimental design was completely randomized blocks, with 30 treatments and three replications, arranged in 5x3x2 factorial outline. The treatments consisted of five N rates: 2, 15, 30, 45 and 60 mg N kg(-1) soil; three sampling times: 23, 40 and 76 days after sowing (DAS) and two crops: common bean and cowpea. The BNF decreased with increase N rates, varying from 81.5% to 55.6% for cowpea, and from 71.9% to 55.1% for common bean. The symbiotic N(2) fixation in cowpea can substitute totally the nitrogen fertilization. The nitrogen absorption from soil is not affected by nitrogen fertilizer rate. The N recovery from fertilizer at 76 DAS was of 60.7% by common bean, and 57.1% by cowpea. The symbiotic association in common bean needs the application of a starting dose (40 kg N ha(-1)) for economically acceptable yields.

Mycovirus in Pseudocercospora griseola, the causal agent of angular leaf spot in common bean

Pseudocercospora griseola (Sacc.) Crous &. Braun is a widespread fungal phytopathogen that is responsible for angular leaf spot in the common bean (Phaseolus vulgaris L.). A number of fungal phytopathogens have been shown to harbour mycoviruses, and this possibility was investigated in populations of Pseudocercospora griseola. The total nucleic acid extracts of 61 fungal isolates were subjected to agarose gel electrophoresis. Small fragments (800-4800 bp) could be identified in 42 of the samples. The presence of dsRNA in isolate Ig838 was confirmed by treatment of total nucleic acid with DNase, RNase A, and nuclease S I. Transmission electron microscopy revealed the presence of viral-like particles 40 nm in diameter in the mycelia of 2 fungal isolates, namely 29-3 and Ig838. The transmission of dsRNA by means of conidia was 100% for isolate 29-3, but there was loss of 1-6 fragments of dsRNA in monosporic colonies of isolate Ig848. Cycloheximide treatment failed to inhibit the mycovirus in isolate 29-3, but proved efficient in the elimination of the 2.2, 2.0, 1.8, 1.2 and 1.0 kb fragments in 2 colonies of isolate Ig848. The occurrence of a mycovirus in Pseudocercospora griseola was demonstrated for the first time in the present study.

Patterns of resistance to angular leaf spot, anthracnose and common bacterial blight in common bean germplasm

Diseases and insect pests are major causes of low yields of common bean (Phaseolus vulgaris L.) in Latin America and Africa. Anthracnose, angular leaf spot and common bacterial blight are widespread foliar diseases of common bean that also infect pods and seeds. One thousand and eighty-two accessions from a common bean core collection from the primary centres of origin were investigated for reaction to these three diseases. Angular leaf spot and common bacterial blight were evaluated in the field at Santander de Quilichao, Colombia, and anthracnose was evaluated in a screenhouse in Popayan, Colombia. By using the 15-group level from a hierarchical clustering procedure, it was found that 7 groups were formed with mainly Andean common bean accessions (Andean gene pool), 7 groups with mainly Middle American accessions (Middle American gene pool), while 1 group contained mixed accessions. Consistent with the theory of co-evolution, it was generally observed that accessions from the Andean gene pool were resistant to Middle American pathogen isolates causing anthracnoxe, while the Middle American accessions were resistant to pathogen isolates from the Andes. Different combinations of resistance patterns were found, and breeders can use this information to select a specific group of accessions on the basis of their need.

Genetic variability in cultivated common bean beyond the two major gene pools

It is generally accepted that two major gene pools exist in cultivated common bean (Phaseolus vulgaris L.), a Middle American and an Andean one. Some evidence, based on unique phaseolin morphotypes and AFLP analysis, suggests that at least one more gene pool exists in cultivated common bean. To investigate this hypothesis, 1072 accessions from a common bean core collection from the primary centres of origin, held at CIAT, were investigated. Various agronomic and morphological attributes (14 categorical and 11 quantitative) were measured. Multivariate analyses, consisting of homogeneity analysis and clustering for categorical data, clustering and ordination techniques for quantitative data and nonlinear principal component analysis for mixed data, were undertaken. The results of most analyses supported the existence of the two major gene pools. However, the analysis of categorical data of protein types showed an additional minor gene pool. The minor gene pool is designated North Andean and includes phaseolin types CH, S and T; lectin types 312, Pr, B and K; and mostly A5, A6 and A4 types alpha-amylase inhibitor. Analysis of the combined categorical data of protein types and some plant categorical data also suggested that some other germplasm with C type phaseolin are distinguished from the major gene pools.

Evaluation of the potential of translocated common cockle for ecological risk assessment studies: bioaccumulation and biomarkers test

Thesis submitted to the Faculdade de Ciências e Tecnologia to obtain the Master’s degree in Environmental Engineering, profile in Ecological Engineering

The trematodes and cestodes of Sorex araneus L. in Valaam Island (Ladoga Lake, USSR). 1

The trematode and the cestode fauna was examined in 50 specimens of common shrews Sorex araneus L. (Insectivora: Soricidae) cillected in Valaam Island, URSS during 1988 and 1989. Two species of Trematoda and seven species of Cestoda were identified; prevalence of infection was as follows: Brachulaemus fulvus (86% ), Rubenstrema exasperatum (4% ), Hymenolepis scutigera (54% ), Neoskrjabinolepis schaldybini (26% ), Vigisolepis spinulosa (4% ), Choanotaenia crassiscolex (86% ), Choanotaenia hepatica (6% ), Dilepis undula (2% ) and Polycercus sp. (2% ).

Bioaccumulation of 210Pb and 210Po in fish tissues in a radioactive naturally enhanced area: the Peníscola marsh (Castelló, Spain)

210Pb and 210Po concentration in fish tissues from Peníscola marsh were analyzed, being this area a radioactive naturally enhanced marsh located in the East coast of Spain. Results showed that 210Po accumulation in tissues could reach values ranging from 28±8 Bq kg-1 in muscle of Cyprinus carpio, to 8558±6378 Bq kg-1 in gut content of Chelon labrosus. On the other hand, 210Pb concentrations ranged from 8±4 Bq kg-1 in muscle of Cyprinus carpio, to 475±481 Bq kg-1 in gut content of Chelon labrosus. Bioaccumulation pattern is generally 210Po&210Pb, except in spine, where more 210Pb than 210Po is accumulated. When comparing our samples to those collected as blanks, individuals from Peníscola marsh showed an enrichment in 210Po and 210Pb in their tissues compared to the blanks. Bioaccumulation factors showed that feeding is the major input route of 210Pb and 210Po into the fish body. Highest values of 210Pb and 210Po concentration in tissues were found on Chelon labrosus and Carassius auratus, being Cyprinus carpio the species with the lowest average values of 210Pb and 210Po accumulation.

Aspectos biológicos de Zabrotes subfasciatus (Bohemann, 1833) (Coleoptera, Bruchidae) em Phaseolus vulgaris L., cv. Carioca (Fabaceae), sob condições de laboratório

Zabrotes subfasciatus is a serious pest of common beans, P. vulgaris L.. In Brazil there are several studies dealing with resistance of bean genotypes to this insect, while other studies have emphasized the utilization of oils and powders from plants to repel their attack. In this paper, fecundity, fertility, pattern of oviposition, life cycle and longevity were evaluated for a Brazilian stock from the Goiás State on P. vulgaris cv. Carioca, at 30ºC and 70% R.H. The mean fecundity was 38 eggs per female and 73% of viability. Egg laying showed an aggregated pattern. Males and females lived an average of 13 and 9 days, respectively. The total life cycle lasted for about 28 days.

Leaf senescence of common bean plants as affected by soil phosphorus supply

Responses of leaf senescence to P supply could constitute adaptive mechanisms for plant growth under P-limiting conditions. The aim of this study was to evaluate the effects of soil P supply on leaf senescence of common bean (Phaseolus vulgaris L.). Eight P levels, ranging from 5 to 640 mg kg-1 P, were applied to pots containing four bean plants of cultivar Carioca in 10 kg of an Oxic Haplustult soil. Attached leaves were counted weekly, abscised leaves were collected every other day, and seeds were harvested at maturity. The number of live leaves increased until 48 days after emergence (DAE) and decreased afterwards, irrespective of applied P levels. At lower applied P levels, the initial increase and the final decrease of leaf number was weak, whereas at higher applied P levels the leaf number increased intensively at the beginning of the growth cycle and decreased strongly after 48 DAE. Dry matter and P accumulated in senesced leaves increased as soil P levels increased until 61 DAE, but differences between P treatments narrowed thereafter. The greatest amounts of dry mass and P deposited by senesced leaves were observed at 48-54 DAE for high P levels, at 62-68 DAE for intermediate P levels and at 69-76 DAE for low P levels. These results indicate that soil P supply did not affect the stage of maximal leaf number and the beginning of leaf senescence of common bean plants, but the stage of greatest deposition of senesced leaves occurred earlier in the growth cycle as the soil P supply was raised.

Seeds enriched with phosphorus and molybdenum improve the contribution of biological nitrogen fixation to common bean as estimated by 15n isotope dilution

Seeds with a high concentration of P or Mo can improve the growth and N accumulation of the common bean (Phaseolus vulgaris L.), but the effect of enriched seeds on biological N2 fixation has not been established yet. This study aimed to evaluate the effect of seeds enriched with P and Mo on growth and biological N2 fixation of the common bean by the 15N isotope dilution technique. An experiment was carried out in pots in a 2 x 3 x 2 x 2 factorial design in randomized blocks with four replications, comprising two levels of soil applied P (0 and 80 mg kg-1), three N sources (without N, inoculated with rhizobia, and mineral N), two seed P concentrations (low and high), and two seed Mo concentrations (low and high). Non-nodulating bean and sorghum were used as non-fixing crops. The substrate was 5.0 kg of a Red Latosol (Oxisol) previously enriched with 15N and mixed with 5.0 kg of sand. Plants were harvested 41 days after emergence. Seeds with high P concentration increased the growth and N in shoots, particularly in inoculated plants at lower applied P levels. Inoculated plants raised from high P seeds showed improved nodulation at both soil P levels. Higher soil P levels increased the percentage of N derived from the atmosphere (%Ndfa) in bean leaves. Inoculation with the selected strains increased the %Ndfa. High seed P increased the %Ndfa in inoculated plants at lower soil P levels. High seed Mo increased the %Ndfa at lower soil P levels in plants that did not receive inoculation or mineral N. It is concluded that high seed P concentration increases the growth, N accumulation and the contribution of the biological N2 fixation in the common bean, particularly in inoculated plants grown at lower soil P availability.

Variability of root traits in common bean genotypes at different levels of phosphorus supply and ontogenetic stages

Selection of common bean (Phaseolus vulgaris L.) cultivars with enhanced root growth would be a strategy for increasing P uptake and grain yield in tropical soils, but the strong plasticity of root traits may compromise their inclusion in breeding programs. The aim of this study was to evaluate the magnitude of the genotypic variability of root traits in common bean plants at two ontogenetic stages and two soil P levels. Twenty-four common bean genotypes, comprising the four growth habits that exist in the species and two wild genotypes, were grown in 4 kg pots at two levels of applied P (20 and 80 mg kg-1) and harvested at the stages of pod setting and early pod filling. Root area and root length were measured by digital image analysis. Significant genotype × P level and genotype × harvest interactions in analysis of variance indicate that the genotypic variation of root traits depended on soil nutrient availability and the stage at which evaluation was made. Genotypes differed for taproot mass, basal and lateral root mass, root area and root length at both P levels and growth stages; differences in specific root area and length were small. Genotypes with growth habits II (upright indeterminate) and III (prostrate indeterminate) showed better adaptation to limited P supply than genotypes of groups I (determinate) and IV (indeterminate climbing). Between the two harvests, genotypes of groups II and III increased the mass of basal and lateral roots by 40 and 50 %, respectively, whereas genotypes of groups I and IV by only 7 and 19 %. Values of the genotypic coefficient of determination, which estimates the proportion of phenotypic variance resulting from genetic effects, were higher at early pod filling than at pod setting. Correlations between shoot mass and root mass, which could indicate indirect selection of root systems via aboveground biomass, were higher at early pod filling than at pod setting. The results indicate that selection for root traits in common bean genotypes should preferentially be performed at the early pod-filling stage.

Water infiltration in an ultisol after cultivation of common bean

Water infiltration in the soil is an important hydrological process that occurs at the interface of the soil-atmosphere system; thus, the soil management practice used has a strong influence on this process. The aim of this study was to evaluate water infiltration in the soil and compare equations for estimating the water infiltration rate in an Ultisol after harvesting common bean (Phaseolus vulgaris L.) under simulated rainfall. Field tests with a rainfall simulator were carried out in three soil management systems: minimum tillage (MT), conventional tillage (CT), and no tillage (NT). In NT, four levels of plant residue on the soil surface were evaluated: 0, 3, 6, and 9 t ha-1. The models of Kostiakov-Lewis, Horton, and Philip were used to estimate the infiltration rate. In the MT system, the final infiltration rate was 54 mm h-1, whereas in the CT and NT systems with up to 3 t ha-1 of plant residue on the soil surface, the rate was near 17 mm h-1. In addition, the results indicated that in the NT system the infiltration rate increased with plant residue coverage greater than 6 t ha-1, i.e., there was a positive correlation between plant cover and the water infiltration rate. The Horton model was the most suitable in representing the water infiltration process in the soil. Therefore, this model can be recommended for estimation of this variable regardless of the soil tillage system used.

Growth and yield of common bean cultivars at two soil phosphorus levels under biological nitrogen fixation

The genotypic differences on growth and yield of common bean (Phaseolus vulgaris L.) in response to P supply were evaluated in a field experiment under biological N2 fixation. Eight cultivars were grown at two levels of applied P (12 and 50 kg ha-1 of P -- P1 and P2 respectively), in randomized block design in factorial arrangement. Vegetative biomass was sampled at three ontogenetic stages. The effects of genotype and phosphorus were significant for most traits, but not the genotype ´ phosphorus interaction. The cultivars presented different patterns of biomass production and nutrient accumulation, particularly on root system. At P1, P accumulation persisted after the beginning of pod filling, and P translocation from roots to shoots was lower. The nodule senescence observed after flowering might have reduced N2 fixation during pod filling. The responses of vegetative growth to the higher P supply did not reflect with the same magnitude on yield, which increased only 6% at P2; hence the harvest index was lower at P2. The cultivars with highest yields also presented lower grain P concentrations. A sub-optimal supply of N could have limited the expression of the yield potential of cultivars, reducing the genotypic variability of responses to P levels.